These results were likely driven by outliers with extreme baseline viral loads. When considering subjects with baseline cycle threshold values of 20–30, STX-3 showed a significant (p = 0.016) 2.01 log10 reduction. The proportion of subjects that turned negative by the end of treatment (day 5) was significantly higher in the STX-3 group than in controls, suggesting a shorter virus clearance time. STX was safe and well-tolerated. In the in vitro study, ≥99.9% reduction in titers against common respiratory viruses was observed. STX is a safe device with large virucidal spectrum and may reduce viral loads in mild COVID-19 patients.Sentinox (STX) is an acid-oxidizing solution containing hypochlorous acid in spray whose virucidal activity against SARS-CoV-2 has been demonstrated. In this paper, results of a randomized controlled trial (RCT) on the efficacy of STX in reducing viral load in mild COVID-19 patients (NCT04909996) and a complementary in vitro study on its activity against different respiratory viruses are reported. In the RCT, 57 patients were randomized (1:1:1) to receive STX three (STX-3) or five (STX-5) times/day plus standard therapy or standard therapy only (controls). Compared with controls, the log The ongoing COVID-19 pandemic has caused an unprecedented burden on healthcare systems: as of 1 May 2022, more than 500 million confirmed cases and over 6,000,000 deaths have been reported worldwide  and the Compared with systemic drug administration, topical delivery routes have several advantages including increased product bioavailability, reduction of side and off-target effects and improved patient convenience, which leads to increased compliance . The nasThe SARS-CoV-2 viral load in the nasopharynx and nasal cavity is usually higher than in the oropharynx or the saliva ,12. It h10 reduction (p ≤ 0.02) in SARS-CoV-2 load on days 2 and 4, while this decrease (0.98 log10) was not significant (p = 0.069) on day 6 [As shown in a recent Cochrane review on the benefits and harms of nasal sprays and mouthwashes , severalon day 6 . Mouth won day 6 . On the on day 6  have notHypochlorous acid (HClO) is a powerful oxidizing antiviral, antibacterial and antifungal agent that selectively binds with the unsaturated lipid layer, disrupting the integrity of pathogens. Its virucidal effect is driven by chlorination, resulting in the formation of chloramines and nitrogen-centered radicals that damage viral nucleic acids ,21. The Sentinox  is an acid-oxidizing solution containing HClO in a spray formulation. The antimicrobial effect of STX is driven by its unique chemico-physical characteristics: the combination of low pH and high oxidation-reduction potential reinforces the antimicrobial activity of HClO. In particular, the low pH inhibits microbial growth, while the high redox potential destabilizes membrane potential of the microorganisms and facilitates their killing ,24. A prclinicaltrials.gov (NCT04909996) (accessed on 15 April 2022) [This prospective, randomized (1:1:1), controlled, open-label, parallel-group, single-center, phase IV study was conducted in Genoa  between May and November 2021. The study was registered with il 2022) . The stuil 2022)  was adopPotentially eligible participants were prospectively identified from a list of requests for molecular diagnostics of SARS-CoV-2, which was performed at the regional reference laboratory for COVID-19 diagnostics of the San Martino Policlinico Hospital . In particular, community-dwelling subjects aged 18–64 years, residing in the municipality of Genoa, positive for SARS-CoV-2 in reverse transcription real-time polymerase chain reaction (RT-PCR) with cycle threshold (Ct) values ≤ 30 for at least two gene targets (see below), and not having previous positive results on rapid antigen-detecting or nucleic acid amplification tests (NAATs) were potentially eligible. Potentially eligible subjects were first pre-screened by the research staff by telephone, and those willing to participate in the study were visited (day 0) at their home by an expert physician. To be enrolled in the study, subjects had to present with a mild disease and with at least one COVID-19-attributable symptom  arising < three days before the day of potential enrollment. Mild disease was defined according to World Health Organization (WHO) criteria  as symptSTX is an acid-oxidizing sprayable formulation of HClO at 0.005% (50 ppm) in a liquid carrier solution obtained through the proprietary electrolysis process of Tehclo Technology . The solution has a low pH of 2.5–3.0, high oxidation-reduction potential (1000–1200 mV) and an HClO content > 95% of the free chlorine species. Several pre-clinical evaluations (data on file) showed that the device is non-irritating to human skin or mucosa, non-sensitizing, non-mutagenic, non-pyrogenic, not toxic up to 50 mL/kg i.e., non-phototoxic, had no keratolytic effect, and satisfied the requirements of the intramuscular implantation test. Special handling precautions were not required.Currently, the STX nasal spray is certified in Europe as a Class III Medical Device (Certificate Nr. EPT 0477.MDD.21/4200.2) and indicated for irrigation, cleansing and moistening of the nasal cavities for (i), reducing the risk of infections caused by bacteria and viruses, including SARS-CoV-2, by lowering the nasal microbial load; (ii) symptomatic nasal care and (iii) nasal care in case of minor lesions/alterations of the nasal mucosa.STX was delivered in a 50 mL bottle. Each dose consisted of the application of 0.5 mL of STX into each nostril according to the following instructions: (i) tilt the head slightly backwards; (ii) close one nostril and gently insert the nozzle into the other; (iii) gently squeeze the pump five times as you breathe in; (iv) switch to the other nostril and repeat; (v) wait at least 1 min and then blow the nose.Randomization. Participants were randomized (simple randomization) on a 1:1:1 ratio in the following arms: STX-3 (administration of STX three times a day as addon to the standard physician prescribed symptomatic therapy), STX-five (administration of STX five times a day as an addon to the standard therapy) and C . All randomized patients received treatment according to which group they were allocated to. Owing to the study design and procedures, the allocated treatment could not be blinded to both patients and investigators.Patient visits. Eligible patients were visited at their home. Principal study procedures by daily visit are reported in To ensure the treatment compliance, on each treatment day (1–5), the research staff telephoned patients of groups STX-3 and STX-5 10 min before each planned spray administration.Starting from day one, all participants were taken an NP swab in both nostrils. On days one and two, three daily swabs were performed at 8:40 a.m. ± 10 min, 2:40 p.m. ± 10 min and 8:40 p.m. ± 10 min. On subsequent days, only one NP swab was performed at 8:40 a.m. ± 10 min. Before swabbing, subjects were asked to blow the nose. In order to harmonize the swabbing procedure, all research staff members were provided unique instructions and an educational video . Each coFollowing completion of the treatment (days one to five), a total of three follow-up visits on days six, 10 and 21 were performed. At each follow-up visit, both clinical examination and NP swabbing were performed. Moreover, on day six the patient diary was assessed for completeness and was retrieved together with the rest of the investigational product. On the same day and following a physical examination, patients in groups STX-3 and STX-5 were asked to rate the overall tolerability of the investigational product and their satisfaction. This was performed by means of a 10-cm visual analogue scale (VAS) and 5-point  Likert scale, respectively.Reverse transcription real-time polymerase chain reaction (RT-PCR). Each specimen collected was tested by means of both qualitative and quantitative RT-PCR at the regional reference laboratory for COVID-19 diagnostics of the San Martino Policlinico Hospital . First, total RNA was extracted by means of the STARMag Universal Cartridge Kit  on the automated Nimbus IVD  platform according to the manufacturer’s instructions . In part1, 102, 103, 104, and 105 copies/mL of a synthetic viral N1-encoding RNA [Quantitative RT-PCR was then performed in order to quantify the viral load (copies/mL) by using the Quanty COVID-19v2 assay  according to the manufacturer’s instructions . Brieflyding RNA .Independent evaluations ,37 have t0 and td, where d is the day of follow-up. The secondary efficacy endpoint was set in order to compare the time length to negativization between the study arms. As described earlier, subjects were defined as negative according to two different Ct cut-off definitions, namely 40 [The primary endpoint was the efficacy of STX in reducing viral load at any time during the days of treatment (days one to five), as determined by quantitative RT-PCR. The absolute change in viral loads was expressed as the difference between amely 40  and 35 [amely 40 ,34. On ta priori on the basis of a clinically relevant reduction in viral load between groups STX-3/STX-5 and C. The clinically relevant reduction was set to 1.5 log10 copies/mL with a standard deviation (SD) of 1.2 log10 copies/mL. A sample size of 14 patients per group was necessary to achieve an 80% power and Bonferroni-corrected two-tailed α of 0.025 of detecting the prespecified difference in viral loads. Assuming a dropout rate of 25%, the final sample size was 57 (19 per group) patients.The sample size was determined All analyses were performed according to the intention-to-treat (ITT) principle. Per-protocol (PP) population analysis was also performed to determine the extent to which the missing data might have influenced the results. The overall effect of STX spray was also verified by pooling the two treatment arms.10 (copies/mL + 1) to account for natural zeros. Continuous variables were expressed as means ± SDs or medians with ranges. Categorical variables were expressed as proportions. A Fisher-Freeman-Halton exact test was used to test differences in proportions. The Cochran–Mantel–Haenszel test was used to compare between-group differences in negativization rates and the corresponding effect sizes were expressed as risk ratios (RRs) with 95% confidence intervals (CIs). For the primary endpoint of change in viral loads, treatment effects were estimated by applying mixed linear models, with analysis of co-variance (ANCOVA) methods with type III orthogonal sum of squares. Time point, treatment regimen and time-per-treatment interaction term were set as fixed effects, while the baseline viral load was used as a covariate. The time points were specified as repeated measures. In the matrix model, the compound symmetry for the observations within each patient was assumed.Individual viral loads were transformed as logAll analyses were performed in SAS v9.4 .A total of seven different respiratory viruses were tested; these were obtained from the American Type Culture Collection (ATCC) . These viruses were: influenza A(H1N1) ; influenza B ; RSV A ; human rhinovirus ; human adenovirus type 5 ; human parainfluenza virus type 3 ; seasonal coronavirus .2. On the day of testing, cell cultures were inspected for their integrity and confluency. Virus-specific test media used in the study are reported in Working stocks of viruses were prepared by passaging in MDCK (for both influenza viruses), Hep-2 (for RSV), MRC-5 (for rhinovirus), A-549 (for adenovirus), MDBK (for parainfluenza virus) and WI-38  cell cultures. The stock virus cultures were adjusted to contain 1% fetal bovine serum (FBS). The cells were seeded into multi-well cell culture plates and maintained at 36–38 °C in a humified atmosphere of 5–7% COSTX was used in its commercially available formulation. For the treatment of virus suspension, a 1.80 mL aliquot of the test substance was dispensed into a separate tube and mixed with 200 µL of the virus suspension, vortexed for 10 s and incubated at 35 °C. The exposure time assayed ranged from 15 to 120 s. Immediately after each exposure time, a 100 µL aliquot was removed from each tube and tittered by 1:10 serial dilutions. In parallel, water was tested as the virus control. Neutralization and cytotoxicity controls were also tested to ensure that the virus inactivation did not continue after the pre-specified contact time.The above-described cell lines, which exhibit a cytopathic effect in the presence of corresponding viruses, were used as the indicator cell line in the infectivity assays. Cells cultured in multi-well dishes were inoculated in quadruplicate with 100 µL of the dilutions prepared from the test and control groups. Uninfected indicator cell cultures (controls) were inoculated with test medium alone. The cultures were scored periodically for the presence or absence of cytopathic effect, cytotoxicity and viability.10 of the median tissue culture infectious dose (TCID50), and computed by applying the Spearman-Karber’s method [10 reduction value (LRV) of the STX vs. negative control was calculated.Infectivity and cytotoxicity titers were expressed as −logs method . Finallyn = 18), STX-5 (n = 20) and C (n = 19). There were three consent withdrawals (one in each group). A total of five patients (2 in group STX-5 and three in group C) terminated the participation earlier due to the worsening of clinical conditions. Therefore, the final ITT population included 54  patients, while the PP population included 49 patients   .p = 0.40), the viral load at visit 0 was higher in the STX-5 group than in the STX-3 and C groups (C groups .p < 0.0001) decay in viral loads independently of the ITT study arm. The overall effect of treatment was not significantly (p = 0.95) different from group C. However, though non-significant, by the end of treatment on day five, patients in group STX-3 showed a 1.02  log10 reduction in viral load, as compared with group C. The corresponding log10 reduction in the STX-5 group was 0.18 . The effects of viral load at screening (p = 0.26) and time-per-treatment interaction (p = 0.59) were not statistically significant. Similar results were observed in the PP population , and all of these had high viral loads (Ct < 20). For this reason, a stratified analysis was conducted by dividing subjects on the basis of Ct values at screening into two categories, namely medium (Ct 20–30) and high (Ct < 20) viral loads. However, it was possible to conduct the inferential analysis only for medium viral load samples, as the number high viral load samples was insufficient. As shown in n = 39, 13 in either group), the efficacy of STX was more pronounced in both treatment arms. In particular, on the last day of treatment (day five), compared with group C, subjects in the STX-3 group showed a significant (p = 0.016) 2.01 (95% CI: 0.37–3.65) log10 reduction in viral loads. The corresponding reduction  in the STX-5 group was not statistically significant (p = 0.53).A visual inspection of the distribution of viral loads at For the secondary efficacy endpoint, the study groups were compared in terms of negativization by using two different cut-offs. As shown in p > 0.99). The most frequent adverse events were cough and sore throat (10.5% each). A total of 4 (21.1%) AEs were classified as serious , but were judged unrelated to the study treatment. All serious AEs were associated with a disease progression to moderate-to-severe pneumonia. Notably, the frequency of severe adverse events leading to hospitalization due to disease progression in the treatment groups was about six times lower (2.8% vs. 16.7%) than in the control group.During the whole follow-up period, a total of 19 AEs were recorded. Of these, only one (5.3%) AE  was judged to be probably related to the study treatment. As shown in For what concerns the tolerability measured on a 1-to-10 VAS, the median scores were 10 in all study arms. Analogously, no between-group difference in patient satisfaction was observed: 94.1%, 100% and 100% of participants in groups STX-3, STX-5 and C were satisfied or very satisfied with treatment.10). In all experiments, the neutralization control  indicated that the test substance was neutralized at ≤1.50 log10. LRVs following exposure to the STX solution are reported in Cytotoxicity was not observed for all viruses tested and any dilution tested (≤1.50 log10 reduction (approximately 90% relative reduction) in SARS-CoV-2 concentration; this decrease was not statistically significant, likely for the reasons described below. On the other hand, when influential outliers were removed, participants in group STX-3 showed a significant viral load reduction of 2.01 log10 copies/mL. Analogously, treatment with STX was generally associated with a shorter time to negativization, which is essential to limit the probability of spreading the virus. The STX spray was safe and well-tolerated in both treatment arms. In the complimentary in vitro study, we also demonstrated a high level of virucidal activity of the STX solution against a variety of other than SARS-CoV-2 respiratory pathogens including influenza A and B, RSV, rhinovirus, adenovirus, parainfluenza virus and seasonal coronavirus. These pathogens are responsible for an important clinical and socioeconomic burden worldwide [To our knowledge, this is the first RCT on the efficacy of a HClO-based solution in reducing the SARS-CoV-2 viral load in patients with mild COVID-19 disease. In the primary analysis, compared with the control group, subjects treated with STX showed a 0.5–1 logorldwide .S. epidermidis, S. pneumoniae, K. pneumoniae, S. aureus) culture positivity rate among adult patients treated with HClO for four weeks were substantially higher (59% vs. 33%) than in those receiving placebo; however, like in the present study and owing to a small sample size, this difference did not reach an α < 0.05 [Besides COVID-19, the efficacy of low-concentration HClO-based nasal irrigations has been evaluated in some RCTs carried out mainly in the field of otorhinolaryngology. In particular, it has been found to be effective against some clinical endpoints relative to chronic rhinosinusitis refractory to medical therapy  or afterα < 0.05 . The preα < 0.05 ,21,22.Despite the observed reduction in viral loads, the primary study endpoint was not met. A subsequent exploratory analysis identified some likely drivers of this finding. Thus, we observed a very high variability in terms of initial viral loads, whose baseline levels differed by approximately five orders of magnitude. This difference reflects both within- and between-subject variations in the viral load curve . On the Notably, three of 18 patients (16.7%) in the control group terminated the trial early as a result of clinical worsening that resulted in hospitalization (two patients were admitted to the intensive care unit). By contrast, considering both treatment groups, only one out of 36 (2.8%) patients reported a severe AE leading to hospitalization due to disease progression. Although the outcome of the clinical progression was not pre-specified, it deserves further investigation. It is indeed plausible that the reduction in viral loads in the upper respiratory tract may reduce and/or slow down virus propagation to the lower respiratory tract.ρ = −0.72–−0.92, depending on the gene target); moreover, most NP samples with Ct > 35 showed viral loads < 5 log10 copies/mL [10 copies/mL is very low. In this regard, our study demonstrated that treatment with the STX spray was associated with a shorter time to negativization, which is essential to reduce the probability of virus transmission.The main strength of this RCT is the two-step approach for the SARS-CoV-2 molecular diagnostics adopted, in which both qualitative (for virus detection) and quantitative  RT-PCR was used. This approach is highly recommended for studies focusing on viral kinetics . Indeed,opies/mL . In a reopies/mL , it has The present RCT suffers from some shortcomings as well. First of all, as the study was designed in a period when little data on SARS-CoV-2 viral kinetics were available and different viral strains were circulating, the assumed statistical dispersion of individual viral loads was underestimated. Second, despite our efforts to uniform the NP swabbing procedure, some within- and between-operator heterogeneity in performance is likely. These variations may be further driven by between-patient differences in nasal anatomy, potentially swollen nasal mucosa or earlier retraction of an incompletely saturated mucus swab due to the patient’s discomfort . Third, 10 copies/mL in subjects with medium viral loads, which are typical for subjects with mild-to-moderate COVID-19 who usually require only outpatient medical attention. Following appropriate counselling performed by general practitioners (GPs), outpatients may therefore be considered the primary target for STX. Furthermore, throughout the whole follow-up period, no safety concerns emerged, confirming its current designation as an over-the-counter medical device. The in vitro study demonstrated a high level of virucidal activity of the STX spray against a variety of respiratory viruses, including influenza and RSV. These promising results support future larger-scale clinical studies in order to assess whether the STX spray is also effective in the primary prevention of both symptomatic and asymptomatic SARS-CoV-2, influenza, RSV and other acute respiratory infections in the at-risk population. These pragmatic trials may, for example, compare the incidence of clinically diagnosed influenza-like illness (with a subsequent molecular diagnostics of the causative agent) between the cohorts of treated and non-treated subjects. Analogously, future research may investigate and compare preventive and treatment effects of different pharmaceutical forms and/or administration routes  of the low-concentration HClO-based solutions.In conclusion, owing to the high variability of baseline viral loads, the primary endpoint of this RCT was not met, the STX spray self-administered three times a day significantly reduced the viral load by about 2 log


Autism spectrum disorder (ASD) is a neurodevelopmental disorder. Abnormal lipid metabolism has been suggested to contribute to its pathogenesis. Further exploration of its underlying biochemical mechanisms is needed. In a search for reliable biomarkers for the pathophysiology of ASD, hippocampal tissues from the ASD model BTBR T+ Itpr3tf/J (BTBR) mice and C57BL/6J mice were analyzed, using four-dimensional (4D) label-free proteomic analysis and bioinformatics analysis. Differentially expressed proteins were significantly enriched in lipid metabolic pathways. Among them, apolipoprotein A-I (ApoA-I) is a hub protein and its expression was significantly higher in the BTBR mice. The investigation of protein levels (using Western blotting) also confirmed this observation. Furthermore, expressions of SphK2 and S1P in the ApoA-I pathway both increased. Using the SphK inhibitor (SKI-II), ASD core phenotype and phenotype-related protein levels of P-CREB, P-CaMKII, and GAD1 were improved, as shown via behavioral and molecular biology experiments. Moreover, by using SKI-II, we found proteins related to the development and function of neuron synapses, including ERK, caspase-3, Bax, Bcl-2, CDK5 and KCNQ2 in BTBR mice, whose levels were restored to protein levels comparable to those in the controls. Elucidating the possible mechanism of ApoA-I in ASD-associated phenotypes will provide new ideas for studies on the etiology of ASD. Autism spectrum disorder (ASD) is a group of neurodevelopmental diseases that manifests in early childhood and is characterized by social communication disorders, repetitive behaviors, and restricted interests. Patients may also experience cognitive impairment, sensory impairment, anxiety, and depression . The preThe etiology and pathogenesis of ASD remain unknown, and studies have focused on genomics, metabolomics , and imaDue to the availability of limited tissue samples from patients with ASD, some studies have used mouse models that express traits relevant to autism to gain mechanistic insight. It has been shown that among these models, the BTBR T+ Itpr3tf/J (BTBR) mouse model shows most of the behaviors that are the core clinical manifestations in patients with ASD ,13. ThesProteomics is a valuable tool for identifying key molecules and pathways involved in various physiological and pathological processes . It has The experimental design of this study is illustrated in p < 0.05), of which the expression levels of 78 proteins were increased and those of 46 proteins were reduced in the hippocampal tissue  and C57 mice  were subjected to 4D label-free proteomic analysis for differential protein screening. We focused on 124 proteins with significant changes in their expression levels  and Clusters of orthologous groups for eukaryotic complete genomes (KOG) databases to analyze the differentially expressed proteins involved in functional modulation in BTBR mice. According to GO functional classification, most proteins were enriched in cellular and metabolic processes d. ProteiA GO/Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis was used to analyze differentially expressed proteins . Figure V.11.0, https://string-db.org/ accessed on 16 October 2022) protein interaction database to conduct an interaction analysis of all the differentially expressed proteins to identify key regulatory proteins. The top 50 high-confidence proteins in the interaction network (score > 0.7) were retained for visualization (We used the STRING (version lization a. In thelization b. When ep < 0.0008).To further validate the bioinformatic analysis results, Western blotting was used to analyze the protein levels of ApoA-I. The results revealed that ApoA-I protein expression was significantly higher in the hippocampal tissues of BTBR mice than in the controls . SphK2 protein expression was higher in the hippocampal tissues of BTBR mice than in the controls (p = 0.0036). The distribution of S1P in the hippocampus (p = 0.0088) and serum (p = 0.0299) was higher in the BTBR group than that in the control group. According to the literature, ApoA-I can increase S1P release by modulating SphK activity . Subsequp = 0.0282) and SphK2 (p = 0.0073), but not SphK1 (p = 0.7750). S1P levels in the hippocampus (p = 0.0019) and serum (p = 0.0122) were dramatically reduced after the SKI-II intervention.Protein detection revealed that SphK inhibitor (SKI-II) intervention in the BTBR group downregulated ApoA-I ; however, BTBR mice showed social interaction deficits when choosing between the unfamiliar mice and the object (p = 0.2700). The social preference test, which examined whether the experimental mice preferred socially novel or familiar mice, revealed that the BTBR group showed reduced social novelty recognition , which differed from the control group . We further investigated the effect of SKI II on the autistic-like behavioral phenotypes of BTBR mice and found that it improved their impaired social preference .ASD-like behavioral phenotypes of BTBR mice have been reported previously . In the p = 0.0053), greater movement time (p < 0.0001), and more self-grooming (p = 0.0001) than the control group. SKI II reduced self-grooming (p = 0.0101) in BTBR mice. However, SKI II intervention did not affect the distance moved (p = 0.9276) or movement time (p = 0.5278) in BTBR mice.In the anxiety-related behavioral test, the open field and self-grooming tests, the BTBR group displayed greater distance moved (p = 0.0441), whereas there was a significant increase in the passing times in the SKI II treatment groups, compared to that in the BTBR group (p = 0.0441). In summary, blocking the ApoA-I-SphK pathway in BTBR mice improved most of the ASD-related phenotypes in these mice.The Morris water maze test was used to assess the effects of the SKI II on spatial learning and memory. The passing times of mice in the BTBR group were lower than those in the control group (p = 0.0017), phosphorylated CaMKII (P-CaMKII)/CaMKII (p = 0.0444), and anxiety-related moleculeGAD1 (p = 0.0075) were lower in BTBR mice than in the control mice. In the BTBR + SKI II group (p = 0.0441), P-CaMKII/CaMKII (p = 0.0008), and GAD1 (p = 0.0089) in the hippocampus reached levels comparable to those in the controls. Inhibition of the ApoA-I-SphK pathway by SKI II may improve behavioral performance regarding cognition-dominant learning, memory, and anxiety disabilities in ASD model mice.ApoA-I pathway-induced alterations in the hippocampus were reversed by the SphK inhibitor SKI II. The cognition- and spatial learning-related proteins CREB and CaMKII and the anxiety-related protein GAD1 were examined to explore the causes of impaired ASD-associated behavioral performance. Results were confirmed using western blot, which showed no alteration in the total protein levels of CREB and CaMKII in each group; however, the expression of phosphorylated CREB (P-CREB)/CREB , a key MAPK signaling regulator, but there was no difference in the expression of phosphorylated P38(P-P38)/P38  compared with that in the control group. Subsequently, we examined MAPK pathway proteins after SKI II blockade in the BTBR + SKI II group (p < 0.0001), but did not regulate P-P38/P38 . ApoA-I-induced alterations in the MAPK signaling pathways were reversed by the sphingosine kinase inhibitor SKI II.The mitogen-activated protein kinase (MAPK) pathway is responsible for cognition, including learning and memory, and studies have shown that abnormal MAPK pathways are correlated with ASD . In FiguII group d,e. SKI p = 0.0495), pro-apoptotic caspase-3  and Bax  increased, whereas those of the anti-apoptotic protein Bcl-2  decreased in the BTBR group. Compared with the BTBR group, in the BTBR+SKI II group (p = 0.0417), caspase-3 , and Bax  were downregulated, whereas Bcl-2  was upregulated. All aberrantly regulated apoptosis-related proteins in the hippocampus of BTBR mice improved following SKI II intervention.To investigate whether high ApoA-I levels were associated with apoptosis, we measured the expression levels of apoptosis-associated proteins, including caspase-3, Bax, Bcl-2, and cyclin-dependent kinase 5 (CDK5). Compared to the control group, II group a,b, CDK5t test, p = 0.6518), KCND3 , and KCNJ10  were not significantly different between the BTBR and control groups; only KCNQ2 (p = 0.0015) and KCNQ3 (p = 0.0143) expression levels were downregulated in BTBR mice. The potassium channel subunit KCNQ2 predominantly assembles with homologous KCNQ3 to form the “M” channel, which is mainly expressed in the hippocampus and cortex. Surprisingly, after SKI II intervention between the groups, both the protein (p = 0.0387) and mRNA (p = 0.0309) levels of KCNQ2 were upregulated in the BTBR+SKI II group compared to the BTBR group, and yet there were no significant differences in KCNQ3 mRNA (p > 0.9999), or protein levels (p = 0.2279). This demonstrates that the abnormally functioning M channels were mainly mediated by downregulated KCNQ2, which could be rescued by altering ApoA-I. However, the heteropolymerization of KCNQ3 showed no change after the intervention. Abnormal KCNQ2 channel function is strongly associated with ApoA-I-related pathways.Potassium channel dysfunction is relevant to the pathophysiology of ASD, particularly with regard to brain function abnormalities and neuronal excitability alterations . The expASD is a neurodevelopmental disorder of unknown etiology. In this study, we focused on the quantitative proteomic analysis of hippocampal tissue from an ASD model, BTBR mice. We used bioinformatic approaches and screened for ApoA-I, a protein that may have a close relationship with ASD. A targeted ApoA-I study was set up, using molecular biology experiments to validate the above-mentioned observations. The abnormal expression levels of ApoA-I signaling downstream molecules SphK and S1P were also confirmed in this study. The SphK inhibitor (SKI II) was subsequently used as an intervention, and behaviors were observed to verify the effect of ApoA-I-related pathway blockade in ASD and the improvement of ASD-associated core phenotypes. Moreover, the mechanisms of ApoA-I action on ASD were elucidated, which were found to involve cognitive proteins, apoptotic proteins, MAPK signaling pathways, and potassium channels. Neurodevelopmental and neurodegenerative disorders are characterized by abnormal lipid metabolism and accumulation . PatientApoA-I, a high-density lipoprotein (HDL) apolipoprotein, is a critical factor in the formation of HDL in the blood . ApoA-I S1P is produced by SphK type 1 and 2. After the SphK intervention, we examined the behavioral alterations in BTBR mice to explore the ApoA-I downstream pathways, S1P levels, and expression of cognitive- and social ability-linked proteins. In the behavioral analysis, the BTBR group demonstrated a significant loss in social ability, repetitive and stereotyped behaviors, cognitive dysfunction, and anxiety behaviors compared with the control group, which is highly consistent with previous investigations . It is gIn the brain, GAD1 is present in GABAergic neurons. ASD is also accompanied by dysregulation of the GABA energy system. The GABA metabolic pathway is one of the causes of anxiety in VPA-induced ASD models ,34. In a2+/calmodulin-dependent kinase (CaMKII) pathway and its downstream molecule, the cAMP response element binding (CREB) protein, play important roles in learning and memory in the hippocampus [The hippocampus is a critical integration center for learning and memory . Recent pocampus . In a nepocampus . In bothpocampus . The finThe MAPK/ERK signaling pathway is highly relevant to neurodevelopmental disorders, especially ASD . The bloIn the hippocampal tissues of BTBR mice, we found alterations in the pro-apoptotic protein caspase-3, which has been identified as a key mediator of physiological neuronal apoptosis . The celPotassium channels play a key role in the modulation of neuronal excitability and synaptic activity. Studies have shown that genetic mutations in ion channels are involved in ASD pathogenesis . We examBTBR T+ Itpr3tf/J (BTBR) mice were bred at the Jackson Laboratory as a model for ASD. C57BL/6J mice were bred at the Beijing Vital River Laboratory Animal Technology Co., Ltd. To exclude possible changes in social behavior due to unequal confounding of each group, one mouse from each group was placed in a cage in a controlled environment  with food and water available ad libitum. All experimenters were blinded to treatment conditions and experimental groupings.g). Protein concentration was determined using a BCA kit . Regarding peptide length distribution, most of the peptides ranged between 7 and 20 amino acids. Regarding peptide number distribution, in most proteins, more than two peptides were involved. When the proteins were quantified, one peptide per protein-specific peptide (or spectrum) enhanced the accuracy and credibility of the quantification results. Regarding protein coverage distribution, the protein coverage was less than 20% for most of the proteins.Equal amounts of protein from each sample were digested, and the lysis solution was adjusted to the same protein concentration. Samples were lysed by ultrasonography and centrifuged to remove cell debris  is a tool used for bioinformatic data analysis that can be used to comprehensively describe the properties of genes and gene products in organisms. GO has a total of three ontologies, including molecular function, cellular component, and biological process, which explain the biological roles of proteins from different perspectives. The differentially expressed proteins were analyzed using eggNog-Mapper software (version 2.0) based on the EggNoG database. The GO ID was obtained from the annotation result of each protein; after this, the protein was classified based on the cellular component, molecular function, and biological function. A statistical analysis was performed on the distribution of differentially expressed proteins in GO secondary annotations.http://www.ncbi.nlm.nih.gov/COG/) accessed on 7 May 2021, stands for “homologous protein cluster”. COG was divided into two groups, one with prokaryotes and the other with eukaryotes. Clusters of orthologous groups (COG) and KOG were used to define orthologs in prokaryotes and eukaryotes, respectively. By database comparison and analysis, COG/KOG functional classification statistics were performed for differentially expressed proteins.COG, Clusters of Orthologous Groups of proteins  pathways by applying a two-tailed Fisher’s exact test; it was considered significant when the The STRING database was used for analyzing the interaction between differentially expressed proteins. A confidence score of >0.7 (high confidence) was considered as a cut-off value, and the top 50 proteins in the interaction relationship were visualized by using the R package networkD3 to obtain the PPI network. Subsequently, the CytoHubba plugin of the Cytoscape software (version 3.9.0) was used to further screen Hub proteins.SKI II is a sphingosine kinase inhibitor that reduces S1P levels in the serum and brain when given intraperitoneally . Four-weThe experimental environment was kept quiet throughout testing. Before testing, the animals were exposed to the environment for at least 2 days. All of the behaviors were evaluated blindly. Two weeks after SKI II (or vehicle) intervention, the open field behavior test, the three-chamber test, the hair grooming test, and the Morris Water Maze test were performed on the mice. At the end of each test session, 75% alcohol was used to clean the inner wall and bottom of the arena to eliminate the residual urine and odor of the tested mouse. All behaviors were evaluated blindly.The open field analysis was used to measure anxiety-like behavior in mice. The test was performed in a quiet environment using the SMART 3.0 opening experiment system. The experimental field was a 45 cm × 45 cm × 40 cm open box made of black polycarbonate, and a far-infrared camera was placed above the box. Mice were placed in the center of the box for simultaneous imaging and timing. Each mouse was allowed to explore the chamber for 10 min and the trajectory, time, and distance of the mice were recorded.The three-chamber test was used to measure the sociability of the mice. During the habituation phase, mice were placed in the middle of the three-chamber apparatus for 10 min. The three-chamber apparatus is a box with three chambers, each area of the chamber is 20 cm × 40 cm, and the chambers on the left and right sides each contain empty barred cylinders on both sides. During sociability testing, the cylinders were filled with visual and olfactory cues so that the stimulus mice can observe and smell each other. The test mice and stimulus mice were able to directly communicate with each other through the cylinders. When the test animals were within 2 cm of a stimulus mouse in the cylinder, this was considered as evidence of social behavior. The social ability test began after the adaptation phase and the stranger mouse 1 (which the test mice did not recognize) was placed in the cylindrical mouse cage in the left chamber. The empty cage was placed in the right chamber, and the test mice were placed in the central chamber. In addition, the movement and activity time in each box were recorded by the camera for 10 min. This phase tested the preference of the test mice for engaging with the stranger mouse in comparison with the empty cage. Then, the social preference test began and the cage with mouse 1 was switched to the right chamber, while the cage with mouse 2 was placed in the left chamber. The movement and activity time of the test mouse were recorded by a camera for 10 min. This phase tested the preference of the test mice for engaging with the new, stranger mouse in comparison with the familiar mouse.Experiments were performed to test the level of stereotyped behavior in mice. Throughout the grooming chain, various forms of strokes and licks were all marked as grooming. Mice were placed into an empty standard cage for 10 min, and subsequently recorded. The duration spent grooming over 10 min was calculated during which the environment remained quiet.The Morris water maze test was used to assess cognitive function. Mice were tested at P42–P46 and the experimental equipment used included the WMT-100 Morris water maze automatic analysis system, with a 160 cm diameter tank carrying a far-infrared camera. The sink was divided into four quadrants, and an 8cm × 8cm platform was placed on the third quadrant. The sink was filled with an opaque liquid at a temperature of 22 ± 1 °C. The liquid was made opaque with non-toxic white paint. The water surface was above the platform by 1–2 cm. The experiment included training on days 1–4, and the training was performed twice daily. A training trial was completed when the mouse found a platform within 60 s and after the trial, the mouse remained on the platform for 30 s after climbing onto it. On day 5, spatial exploration capability was tested. The platform was removed and the mice were placed in the first quadrant to record the number of times they crossed the original platform position within the 60 s. Mice with cognitive deficits should cross the platform less often during the exploration experiments.Differences in S1P levels in mouse hippocampal tissues and serum were determined by the S1P Elisa Kit . The experimental operation was performed according to the manufacturer’s instructions. Standard curves were generated to calculate the concentration value of each sample based on the sample protein content (μ mol S1P/mg protein), and the concentrations of S1P in the serum and hippocampal tissues were expressed in molar quantities.® Premix Ex TaqTM II . The amplification and melting curves of real-time qPCR were confirmed after the reaction. Each sample’s relative expression level was calculated using the 2−∆∆CT method. The primer sequences used in this experiment are listed in Total RNA was extracted from the hippocampus of the mice using RNAiso Plus , isopropanol, and 75% ethanol. RNase-free water was finally added to dissolve the RNA. The Nanodrop 2000 spectrophotometer was used to determine the concentration and purity of the total RNA with the OD260/OD280 ratio of >1.9 as the criteria. For reverse transcription, the PrimeScriptTMRT Reagent Kit with gDNA Eraser  was used according to the instructions provided by the manufacturer. Real-time RT-qPCR analysis was performed in a LightCycler480 System with the TB GreenA total of 30 μg of protein was loaded onto and separated on precast gels, transferred to polyvinylidene fluoride (PVDF) membranes, and probed with antibodies. Horseradish was used to detect the immunoreactive bands and peroxidase-conjugated secondary antibodies were visualized with enhancement. Bands were detected and quantified using Image-Pro Plus software by chemiluminescence. The experiments were repeated at least three times. The antibodies used in this experiment were listed as follows: ApoA-I , SphK1 , SphK2 , phospho-p38 MAPK , p38 MAPK , phospho-p44/42 MAPK , p44/42 MAPK (Erk1/2) , CDK5 (D1F7M) , GAD1 (D1F2M) , Bax (D3R2M) , Bcl-2 (D17C4) , caspase-3 , phospho-CaMKII (Thr286) (D21E4) , CaMKII-α (D10C11) , CREB , phospho-CREB , KCNQ2 , KCNQ3 , KCNQ5  β-actin , β-tubulin  and GAPDH .p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001; **** p < 0.0001). The Shapiro–Wilk test was used to assess the normality of the data distribution. The Student’s t test was used for comparisons between the two groups. Analyses using one-way ANOVA were used for comparisons between the three groups. If there was a violation of variance homogeneity, Welch’s correction was applied. If there was a violation of normality criteria in the normality and lognormality tests, non-parametric tests were used, and the Kruskal–Wallis test was used for between-group comparisons.Statistical analysis of the data was performed with SPSS 26 . The data were described as mean ± SEM, with a significance threshold of α = 0.05 (NS To obtain unbiased data, only the investigator administering the treatment was unblinded to the treatment information. The investigators that performed the behavioral analysis and biochemical analysis were blinded to the treatment grouping.In ASD pathogenesis, lipid metabolism may play an important role , but the


COVID-19 has caused a global pandemic and the death toll is increasing. With the coronavirus continuously mutating, Omicron has replaced Delta as the most widely reported variant in the world. Studies have shown that the plasma of some vaccinated people does not neutralize the Omicron variant. However, further studies are needed to determine whether plasma neutralizes Omicron after one- or two-dose vaccine in patients who have recovered from infection with the original strain.The pseudovirus neutralization assays were performed on 64 plasma samples of convalescent COVID-19 patients, which were divided into pre-vaccination group, one-dose vaccinated group and two-dose vaccinated group.In the three groups, there were significant reductions of sera neutralizing activity from WT to Delta variant (B.1.617.2), and from WT to Omicron variant (B.1.1.529) (ps<0.001), but the difference between Delta and Omicron variants were not significant (p>0.05). The average neutralization of the Omicron variant showed a significant difference between pre-vaccination and two-dose vaccinated convalescent individuals (p<0.01).Among the 64 plasma samples of COVID-19 convalescents, whether vaccinated or not, Omicron (B.1.1.529) escaped the neutralizing antibodies, with a significantly decreased neutralization activity compared to WT. And two-dose of vaccine could significantly raise the average neutralization of Omicron in convalescent individuals. In the past two years, the COVID-19 pandemic has produced wave after wave of SARS-CoV-2 mutants, which beat the early variants in showing partial resistance to neutralizing antibodies induced by natural infection and vaccination. The earliest mutants carried unimodal mutation D614G, which provided an advantage for transmission, and quickly replaced the ancestral virus as the main pandemic variant before May 2020 .The newly emerged SARS-CoV-2 Omicron variant, which was first discovered in South Africa in November 2021, presents a different scenario and causes major concerns , 5. It wWilfredo et al. evaluated the neutralizing immune effect of antibodies induced by mRNA-1273 and BNT162b vaccines against SARS-CoV-2 Omicron mutants and detected no neutralizing antibodies against Omicron in most vaccinated people  according to the instructions. Microporous plates were precoated with SARS-CoV-2 RBD protein. The samples were diluted and added to microporous plates and incubated at 37°C for 1h. The microporous plates were washed for five times. The horseradish peroxidase labeled mouse-anti-human IgG monoclonal antibody was added and incubated at 37°C for 30mins. After incubation, the plate was washed five times, substrate buffer containing hydrogen peroxide and TMB was added to the wells, incubating at room temperature for 15mins. Then the termination solution was added, and a spectrophotometer was used to detect the OD value of the wells under dual-wavelength excitation light of 450nm and 630nm. The antibody titer was obtained by comparing the OD value with the standard substance, which was a monoclonal antibody with a defined titer (1280) and provided in the kit.2.Vero cells (provided by Nanjing Vazyme Biotechnology Co.) derived from the kidneys of normal adult African green monkeys that stably expressing endogenous ACE2. Cells were cultured with DMEM (1%(v/v) antibiotics, 10% (v/v) FBS) at 37°C, supplied with 5% CO2. Plasma from healthy donors before the COVID-19 outbreak were included as negative controls. And negative control wells were supplied with 100μL DMEM (1%(v/v) antibiotics, 10% (v/v) FBS). Positive control  wells were supplied with 100μL DMEM. Pre-mixed Vero cells  were added to all wells, and the 96-well plates were incubated for 24h at 37°C, supplied with 5% CO2. After the incubation, 150μL of supernatants were removed, and 100μL Bio-Lite Luciferase reagent  was added to each well and incubated for 3 mins. After the incubation, luciferase activity was measured using a microplate spectrophotometer. The neutralization titer was calculated by comparing the OD value to the negative and positive control wells.The pseudovirus neutralization assays were performed using Vero cells. Various concentrations of plasma samples  were transferred into 96-well white flat-bottom culture plates, mixed with SARS-CoV-2 pseudoviruses , and incubated for 1h at 37°C, supplied with 5% COStatistical analysis was conducted using GraphPad Prism 8 and SPSS 25. All data in the figures were presented as mean ± SD. The age and gender of three groups were evaluated by chi-square test. Differences of sera neutralizing activity among 3 groups and participants with different times of vaccination were evaluated by one-way ANOVA test. P < 0.05 was considered significant .We included samples from pre-vaccination convalescent individuals (n=25), one-dose vaccinated convalescent individuals (n=17) and two-dose vaccinated convalescent individuals (n=22). All the samples were part of a long-term follow-up COVID-19 convalescents cohort.The median age of the three groups were 37 (range 26-68 years), 45 (range 31-70 years) and 44.5 (range 29-65 years). There were no differences among the 3 groups, neither in age nor in gender Table 1.).Compared to the OD value with the standard substance, antibody titers of all 64 plasma samples were 640 or higher Table 2.The new SARS-CoV-2 viral isolate Omicron B.1.1.529) caused widespread concern because of its high transmissibility and ability to infect the previously exposed or vaccinated individuals , 5. In a29 causedIn this study, we further divided the COVID-19 convalescents into various groups based on their vaccination status. We compared the neutralizing antibody titers after one and two doses of vaccine with that of pre-vaccination among COVID-19 convalescents for the first time. As a result, we found significantly reduced sera neutralizing activity from WT to Delta B.1.617.2) variant, and from WT to Omicron (B.1.1.529) variant. This result was consistent with those of Wanwisa Dejnirattisai et al., Juan Manuel Carreño et al., Jingwen Ai et al. and Sandile Cele et al. 17.2 vari–15. AlthAs the COVID-19 vaccination campaign moves forward, many COVID-19 convalescents had also been vaccinated. We had collected serum samples of such individuals. The results of pseudovirus neutralization assays showed that there was a significant difference between pre-vaccination and two-dose vaccinated convalescent individuals in terms of the neutralizing activity against the Omicron variant. The research of Wanwisa Dejnirattisai et al.  showed aPlasma from patients recovered from COVID-19 infection, namely convalescent plasma, is an explorative treatment with considerable historical evidence in other infectious diseases . Those cThere are several limitations to our study. These include the relatively small sample size which may not represent the neutralization titers in the population, the limited longitudinal data, the lack of data on persistence of neutralization antibody responses after two doses of vaccine, the lack of long-term follow-up, and the lack of live virus neutralization data. However, the current study design allows for a preliminary assessment of the effect of neutralizing antibodies against Omicron variant in convalescents after one and two doses of vaccine. Further studies are needed to confirm the finding.Among the 64 plasma samples of COVID-19 convalescents, whether vaccinated or not, there was a significant decrease in neutralization titer against two kinds of variants compared to WT, which means that Omicron (B.1.1.529) could escaped the neutralizing antibodies. Importantly, two-dose vaccine was found to raise the average neutralization against Omicron in convalescent individuals.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Ethics Committee of the Institute of Blood Transfusion, Chinese Academy of Medical Sciences & Peking Union Medical College. The patients/participants provided their written informed consent to participate in this study.YX, LeL and JW were responsible for plasma collection, antibody titer detection and manuscript-writing. YX also completed data collation and analysis. LiL and ZL contributed to study design, performing the research analysis, and revising the manuscript. LeL, JW and SZ were involved in the discussion about participant recruitment. CL, ZH and YL contacted the convalescents and confirmed their willingness to participate in this study. YZ, WN and YH collected the basic characteristics of the participants. All authors read and approved the final manuscript.This work was supported by the CAMS Innovation Fund for Medical Sciences (CIFMS) , Science & Technology Department of Sichuan Province (2020YFS0583) and the Natural Science Foundation of Hubei Province (2021CFB501).Our gratitude goes to the participants who contributed to this study and Vazyme which support us for the technical assistance.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Surface-enhanced Raman scattering (SERS) has attracted much attention due to its high sensitivity, rapid results, unique spectroscopic fingerprint, and nondestructive data acquisition. In this work, we describe the preparation of flexible CS-ATS-Ag cotton swabs for use in SERS by anchoring silver nanoparticles (AgNPs), as a highly-sensitive SERS material, on cotton swabs (CS) using N-[3-(trimethoxysilyl)propyl] diethylenetriamine (ATS) as the coupling agent. The flexible CS-ATS-Ag cotton swabs exhibited high SERS sensitivity, uniformity and reproducibility as a melamine molecule probe, and the limit of detection was calculated to be 0.2 ppm. A high SERS signal reproducibility was achieved, and the relative standard deviation (RSD) of the melamine peak at 699 cm−1 was approximately 5.01%. Moreover, we successfully developed Chemical analysis App application software; a smartphone was used to convert data and record the results, then the data were geotagged using the GPS feature in the smartphone and uploaded to a central website. The goal of realizing instant transmission, timely processing, high sensitivity, portability and low cost was therefore achieved.It is important to be able to detect melamine  Schematic illustration of the fabrication process of the SERS cotton swabs. Cell phones can be used for data collection and to push data to a website where data is displayed on a map. Since 2007, owing to melamine's high nitrogen content (66% by mass), it has been illegally added to pet food and infant formula, which has attracted much attention. Kidney disease and even death in infants and pets due to ingestion of melamine have been recorded. Considering its potential toxicity, the Codex Alimentarius Commission has set limits for powdered infant formula (1 mg L−1) and other foods and animal feed (2.5 mg L−1).3–5Melamine is a chemical compound that is mainly used for resin production, thermosetting plastic and polymer manufacturing in general, and is widely used as a raw material in industry for producing synthetic polymers.6,7 gas chromatography-tandem mass spectrometry (GC-MS/MS)8,9 and enzyme-linked immunosorbent assay (ELISA),10,11 have been employed for the detection of melamine; however, these methods usually require expensive instruments, and lengthy sample preparation procedures are needed, mainly due to the analyte extraction steps. Surface-enhanced Raman scattering (SERS) has attracted much attention due to its high sensitivity, rapid results, unique spectroscopic fingerprint, and non-destructive analysis for molecule sensing, and has emerged as an up-and-coming technique for chemical and biosensing applications. SERS techniques have unique advantages in various fields, such as biomedicine,12,13 homeland security,14,15 environmental monitoring,16–19 and food safety.20,21 SERS is considered to be one of the most promising and powerful analytical techniques.Recently, some conventional technologies, such as high-performance liquid chromatography (HPLC),22–24 hydrogel,25 polystyrene-block-poly-(ethylene oxide),26 and polydimethylsiloxane27,28 to construct soft and flexible SERS substrates. In recent years, cotton swabs as a support material for the deposition of gold or silver nanoparticles have attracted considerable interest, owing to the low cost of the swabs and their ready accessibility and portability. SERS detection of analytes without the intricate pretreatment process required to obtain the analytes from irregular surfaces is one potential solution. Coupling agents on metal nanoparticles attach to various substrates through their terminal groups. The most commonly- and widely-used coupling agents include thiol-terminated, amine-terminated, alkyl-terminated and phenyl-terminated silane.However, conventional SERS substrates were fabricated on hard supporting substrates such as glass, quartz and silicon. It is difficult to extract the target analytes from the irregular-shaped matrices before SERS detection owing to lack of flexibility of the SERS substrate. In conclusion, a low sample collection efficiency limits application for the detection of analytes on irregular matrices. Therefore, the development of flexible SERS substrates with high SERS activity, uniformity, reproducibility and stability became of increasing interest. For example, metal nanostructures have been deposited onto nanofibers,N1-(3-trimethoxysilylpropyl) diethylenetriamine (ATS) as the coupling agent. The silane coupling agent ATS forms covalent bonds on the CS surface via hydrolysis.29 At the interfaces between CS and silane groups, chemical bonding with –OH occurs on the CS surface. ATS can readily form a monomolecular layer on the CS surface. In the meantime, the presence of three amine groups in ATS can further bind metal ions.30,31 Strong water absorption and flexibility of cotton-based SERS cotton swabs enable it to rapidly capture target molecules from uneven samples by soaking and wiping. These flexible SERS cotton swabs were successfully used to detect melamine at a concentration as low as 0.2 ppm, and the reproducibility of the intensity of the SERS peaks was within 5.01%. More importantly, Chemical analysis App application software was successfully developed and used to convert data and record the results; the data were geotagged using the GPS feature in a smartphone and uploaded to a central website. The goal of realizing instant transmission, timely processing, high sensitivity, portability and low cost was therefore achieved.In this study, we present a simple method for the fabrication of excellent flexibility, highly-sensitive, portable and inexpensive SERS cotton swabs. The SERS cotton swabs with excellent flexibility and high robustness by anchoring AgNPs on cotton fibers using 2.2.1.4, Sigma-Aldrich Co., Ltd.) was used as a reducing agent. N-[3-(trimethoxysilyl)propyl]diethylenetriamine  was used as a coupling agent. Melamine  acted as the target detection molecule. Silver nitrate  was used to reduce AgNPs. Sodium citrate  was used as a protective agent. All chemical reagents used were of analytical grade and were used without further purification. Cotton swabs were purchased from Yuh Shiuans Co., Ltd. The water used in the experiment was ultrapure water (18.2 MΩ).Sodium borohydride (NaBH2.2.The UV-vis absorption spectrum of the silver nanoparticle (AgNP) solution was obtained using a ChromTech CT-8600 instrument . The X-ray diffraction (XRD) patterns were recorded on a Bruker D2 Phaser diffractometer. Scanning electron microscope (SEM) images and the SEM-EDX spectrum of the SERS cotton swabs were recorded using a field-emission SEM . SERS spectra were obtained using a Raman spectrometer  with an excitation wavelength of 532 nm. During detection, the integration duration of the Raman spectra was 10 s.2.3.4) as the reducing agent. An aqueous solution of AgNO3  was added to NaBH4  solution with stirring. The color of the mixed solution changed to brown, and the final color was bright yellow. Then, 1 mL of 0.02 M sodium citrate (TSC) was added to 40 mL of AgNP solution. TSC stabilized the AgNPs in a colloidal state owing to the repulsive force that existed among the particles, and this was maintained by a net negative charge on their surface.32 The above-mentioned procedure was then applied. All AgNP solutions were stored at 4 °C before use.AgNPs were synthesized by a simple chemical reduction method using sodium borohydride (NaBH2.4.Flexible CS-ATS-Ag cotton swabs were fabricated as follows. The cotton swabs were immersed in a 2% (v/v) ATS solution under ultrasonic agitation for 30 min. Then, they were removed from the ATS solution, rinsed with ultrapure water thoroughly, and dried in an oven at 120 °C for 30 min to activate the cotton fibers. The activated CS-ATS cotton swabs were soaked in AgNP solution under ultrasonic agitation for 1 h, following which the AgNP-modified cotton swabs were placed in a vacuum oven and heated at 40 °C for 1 h .2.5.Melamine stock standard solution (1000 ppm) was prepared by dissolving melamine in ultrapure water, and was stored at 4 °C for further use. For sensitivity testing, melamine standard solutions were prepared by subsequent dilution from the stock solution in ultrapure water to attain the following concentrations: 5, 2.5, 1.25, 1, 0.7, 0.5 and 0.2 ppm. The flexible CS-ATS-Ag cotton swabs were immersed in 1 mL of melamine solution at various concentrations for 5 minutes. Finally, Raman spectra of the flexible CS-ATS-Ag cotton swabs were recorded using a Raman spectrometer.For the reproducibility test, the flexible CS-ATS-Ag cotton swabs were immersed in 1 mL of melamine standard solution (5 ppm) for 5 minutes. Raman spectra of seven random spots on the flexible CS-ATS-Ag cotton swabs were recorded and averaged.Two methods were used for stability testing. In the first method, the flexible CS-ATS-Ag cotton swabs were treated with ultrasonication for 30 min, and then taken out to observe whether the silver glue on the surface had fallen off. In the second method, the flexible CS-ATS-Ag cotton swabs were stored in a moisture-proof box for 2 months before testing, and the swabs were then immersed in 1 mL of melamine standard solution (5 ppm) for 5 minutes. The performance of the flexible CS-ATS-Ag cotton swabs after treatment was compared between methods.3.3.1.θ positions 38.2°, 44.2°, 64.4°, 77.5° and 81.6°, corresponding to the (111), (200), (220), (311) and (222) planes of the face-centered cubic (fcc) AgNPs (JCPDS card no. 04-0783).33 In addition, no significant diffraction peaks arising from crystalline impurities were detected, indicating that the product was of a pure cubic Ag phase.34 The crystallite sizes were calculated using the Debye–Scherrer equation:K was the particle shape factor, λ was X-ray wavelength, βhkl was the half-width of the (hkl) reflection, and θ was the Bragg angle corresponding to the (hkl) reflection.35 In this study, we employed the diffraction peak corresponding to the (111) crystal plane to calculate the average grain size (D) of the AgNPs, which was 21.01 nm.The dispersity and morphology of the AgNPs were characterized by UV-vis spectroscopy and SEM. A UV-vis spectrometer was employed to measure the absorbance of the colloidal AgNPs. It can be observed from 36 The signals of C and O may have originated from TSC bound to the surface of the AgNPs, indicating the presence of citrate protection.The EDX profile showed strong signals for C, O and Ag, as shown in 3.2.To demonstrate morphologic changes of the pristine cotton swabs after respective surface modification, SEM characterization of the pristine CS, CS-ATS and flexible CS-ATS-Ag cotton swabs was performed, as shown in 3.3.−1, 678 cm−1 and 983 cm−1 were observed. The most prominent peak at 678 cm−1 was assigned to the ring-breathing II mode of the C atoms of melamine. The second most prominent peak at 983 cm−1 was assigned to the ring-breathing I mode of the triazine ring of melamine.1−1, to evaluate the SERS enhancement effects of the two different types of cotton swab. The pristine CS showed no Raman signal. The SERS signal of melamine obtained from the flexible CS-ATS-Ag cotton swabs after AgNP modification confirmed that the CS-ATS-Ag cotton swab substrate was prepared successfully. The Raman spectrum of the flexible CS-ATS-Ag cotton swabs was obviously changed as compared with the solid Raman spectrum of melamine. The feature peak of melamine solution on the flexible CS-ATS-Ag cotton swabs at 699 cm−1 shifted nearly 21 cm−1 relative to the peak (678 cm−1) in the Raman spectrum of solid melamine. This may be attributed to the effect of the flexible CS-ATS-Ag cotton swab substrate of AgNPs. After mixing with colloidal AgNPs, the feature peak of melamine was red-shifted to 699 cm−1 in the Raman spectrum. It was speculated that the enhancement factor was directly related to both surface plasmon resonance (SPR) and chemical molecular enhancement via charge transfer between the AgNP surface and the adsorbate.373.4.−1 in the SERS spectra of melamine was chosen as the feature band, which was attributed to the unique ring-breathing II mode of the C atoms of melamine.38 To further verify the linear relationship in quantitative detection, three random points were selected on each substrate to record a SERS spectrum. The intensity of the characteristic peak at 699 cm−1 was plotted on the ordinate in order to analyze its linear relationship with the concentration of melamine. It can be seen that the intensity of the representative peak of melamine declined monotonously with the decrease in melamine concentration. In addition, a close linear relationship existed between the intensity of the SERS peak at 699 cm−1 and the melamine concentration, as shown in y = 479.5 + 1548x , which provided a calibration for the quantitative detection of melamine. The limit of detection for melamine using the flexible CS-ATS-Ag cotton swabs was 0.2 ppm, which was below the detection limit that is legislated by the European Union (EU) and the Food & Drug Administration (FDA). In addition, The SERS spectra of melamine at concentrations ranging from 0.20 ppm to 5.00 ppm are presented in 3.5.−1 , can be wirelessly transmitted to a central website and made available to the relevant authorities and the general public. The goal of realizing instant data transmission and timely processing was therefore achieved.We successfully developed Chemical analysis App application software, using Android studio programming language. Our App has four primary functions: (i) conversion of data, (ii) GPS positioning information query, (iii) weather information query, and (iv) test result reporting and transmission. A schematic of our proposed process is presented in 4.i.e., position, weather, image, description and detection results) to food safety officers, providing critical data for policy-makers and the general public. The goal of realizing instant transmission, timely processing, high sensitivity, portability and low cost was therefore achieved.The results showed that the flexible CS-ATS-Ag cotton swabs exhibited an excellent SERS performance, with a detection limit for melamine of 0.2 ppm, and had a high reproducibility, the intensity variation of the SERS peaks being within 5.01%. The flexible CS-ATS-Ag cotton swabs achieved rapid and timely SERS detection of melamine, not only by swabbing of a solid surface, but also by adsorption in solution. More importantly, the flexible CS-ATS-Ag cotton swabs are disposable and portable. Further, our Chemical analysis App transmits test results to a central website, and communicates detection information (The authors declare that there is no conflict of interest in this paper.


PU.1 gene is influenced by repression via DNA methylation, as well as other epigenetic factors, such as those related to progenitor maturation status, which is modulated by the transcription factor Myeloblastosis oncogene (MYB). In this work, we show that combinatorial treatment of acute myeloid leukemia (AML) cells with DNA methylation inhibitors (5-Azacytidine), MYB inhibitors (Celastrol), and anti-miR-155 (AM155) ideally leads to overproduction of PU.1. We also show that PU.1 reactivation can be compensated by miR-155 and that only a combined approach leads to sustained PU.1 derepression, even at the protein level. The triple effect on increasing PU.1 levels in myeloblasts stimulates the myeloid transcriptional program while inhibiting cell survival and proliferation, leading to partial leukemic differentiation.The transcription factor PU.1  is a key regulator of hematopoiesis, whose level is influenced by transcription through its enhancers and its post-transcriptional degradation via microRNA-155 (miR-155). The degree of transcriptional regulation of the  PU.1 gene very efficiently. Previous research in mouse models has shown that a reduction in PU.1 levels represents a situation where there is a progressive block in differentiation and the development of disease that is similar to MPN, even with a propensity to develop AML [PU.1 gene led to the expansion of granulocyte–macrophage (GM) progenitor cells with impaired differentiation potential [One of the hallmarks of myeloid leukemia is the progressive accumulation of clonal precursors that are prevented from developing into normal mature cells. Among the myeloid malignancies, there are chronic progressive diseases such as myeloproliferative neoplasias (MPN) and myelodysplastic syndromes (MDS), which progressively accumulate additional driver mutations and progress to AML. One strategy to inhibit leukemia is the induction of leukemic cell differentiation, a concept that involves therapeutically inducing (often by manipulating transcription factors levels) differentiation and simultaneously arresting division, which ultimately alters the aggressive behavior of the leukemia and normalizes the development of functional blood cells . Normal elop AML . In a siotential , which potential . The regotential ,6,7.PU.1 gene is relatively complexly regulated at the level of several enhancers and at a magnitude of several kb prior to the start of transcription. The deletion of a particular enhancer called the URE (upstream regulatory element), which attracts a number of regulatory transcription factors, disrupts PU.1 expression, such that the URE is unable to associate with and effectively stimulate the PU.1 promoter [The promoter . In addipromoter . Anotherpromoter  and playpromoter . Anotherpromoter . We havepromoter ,14. At tpromoter .PU.1 gene and post-transcriptionally. Our previous work showed that miR-155 is under the influence of the oncogenic transcription factor MYB [Previous studies indicated that while the deletion of PU.1 expression abolishes myeloid development, a reduction in PU.1 levels has an apparent leukemia-promoting effect . The patctor MYB . The inhctor MYB . CEL is ure/ure or control age-matched mice (ure/ure mice (ure/ure progenitors induced by medium-term CEL therapy represented the early myeloid Mac1+c-Kit+ progenitors and late monocyte–granulocytic (Mac1+Gr1+) precursors  affects leukemic differentiation by regulating PU.1 levels. To do this, we used a hypomorphic PU.1 model of AML/MPN in mice , and trehed mice A. As exphed mice A, right.hed mice B, but thhed mice B. The uphed mice . CEL theure mice , but we ure mice E and bloure mice E, the meure mice F. This secursors G. To tesure mice G. In conby AM155 G.ure/ure hypomorphic mutant, the level of PU.1 expression is low [PU.1 locus in AML cells and controls were relatively low. As expected, the mRNA for PU.1 was induced after AZA treatment, while the miR-155, Myb, and MYC proto-oncogene (Myc) levels were reciprocally reduced  to compare the effects of AZA on myeloid progenitors. The stimulatory effects of AZA or G-CSF on PU.1 and its target mRNA levels were comparable  into lethally irradiated recipient mice expressing Cd45.1. The AML engraftment of Cd45.2 donor cells lasted for the next 10 weeks and accounted for more than 60% of the BM hematopoiesis  and NB4 having a relatively smaller hypermethylated region (−14.8 kb to +18.7 kb)  with 50% effect (using the WST1 assay) for CEL and AZA in two human AML cell lines . In the case of AM155, we observed that there was no cytotoxic effect at any level. We used the WST1 assay to test the effects of the triple combination of the selected agents. First, we combined CEL and AZA at different concentrations and observed that the two modalities synergistically elevated PU.1 mRNA . Next, w18.7 kb) B. The 3-18.7 kb) B. Thus, To investigate whether combined treatment with AZA, CEL, and AM155 can inhibit tumor growth in vivo, we used an in vivo methodology to monitor tumor growth in cell-line-derived xenograft (CDX) mice enabled via the intravital detection of a luciferase signal ectopically expressed in tumor cells engrafted within the immunodeficient (non-obese diabetic/severe combined immunodeficiency) NOD/SCID gamma (NSGS) mice. As expected, we observed the engraftment of the tumor tissue from transplanted OCI-M2 cells and verified that tumors derived from CDX mice exhibit the typical cytology of the OCI-M2 cells used for transplantation, i.e., they exhibit typical features of myeloblasts with fine chromatin and bulky nuclei, and we also confirmed via flow cytometry the concordance of their surface antigens . Tumors visualized via luciferase detection from tumor cells showed intraosseous localization and typically linearly expanding growth. Furthermore, we applied the therapy regimen of AZA (100 μg/mouse) and CEL (100 μg/mouse) +/− AM155  3 times a week i.p. compared to vehicle-treated mice. Luciferase detection was performed once a week. From the data involving two groups of mice , we observed statistically significant reductions in tumor mass . The lucOur previous work showed that the deregulation of miR-155 decreased PU.1 levels and that the activation of MYB led to increased aggressiveness of AML . TherefoMiR155hg). Additionally, PU.1 directly controls the expression of three other miRs  through the dynamic occupancy of binding sites in regulatory chromatin regions adjacent to their genomic coding loci [MiR155hg promoter, the mature miR-155 reduces PU.1 protein levels. Conversely, the upregulation of Myb leads to a silencing of PU.1 expression, and similarly PU.1 is one of the repressors of Myb mRNA transcription. Thus, in order to be able to block this nicely matching system, it is important to pull several ends at the same time. Specifically, one must block miR-155 or Myb production and at the same time unblock the repressive action of chromatin in the PU.1 gene region. Therefore, the effects of AZA were utilized in PU.1 derepression, complementing the inhibition of MYB and at the same time inhibiting miR-155.The compensatory role of miR-155, which likely occurs when the progenitor cell takes the path of differentiation, is supported by work suggesting that the upregulation of PU.1 can directly induce the transcription of the host gene for miR-155  has synergistic cell cycle blocking and proapoptotic effects through other miR-155 targets besides PU.1. However, it is known that ectopic and strong overproduction of PU.1 in AML blasts can be associated with both cell cycle arrest and apoptosis, as shown after retroviral transduction with a vector carrying PU.1, which together document the so-called restored partial myeloblast differentiation [One of the key questions regarding ntiation . On the ntiation , so it iThe question remains of how and whether the strategy of leukemic differentiation will be applied in the clinic, and furthermore whether the concept used in our paper can be applied in whole or in part to the relatively broad and heterogeneous field of MDS and AML. There already exist strategies in use today that fit the principles we have presented. An example is a peptidomimetic inhibitor that blocks the formation of the complex between MYB and CREB-binding protein (CBP/p300), which ultimately leads to the inhibition of the MYB targets—MYC and B cell leukemia/lymphoma 2 (BCL2) . Since Mplease note that OCI-M2 is a completely different line from the similarly named OCI-AML2). We also used the NB4 cell line that was derived from a patient with acute promyelocytic leukemia and who displayed the characteristic translocation (15:17) . The WST-1 method for assessing cell proliferation  was used to obtain IC50. Cells were also stained with trypan blue solution. The number of total, live, and dead cells was measured using a Luna IITM Automated Cell Counter .Cell lines: The OCI-M2 cell line was originally derived from a 56-year-old MDS-EB2 patient in transition to AML  Ly5.2 (CD45.2) mice were transplanted into lethally irradiated (7.5 Gy) adult (8 weeks) C57BL/6J Ly5.1 control recipients. At the post-transplantation stage, the mice were tested for the presence of donor-derived cells using a flow cytometric analysis for CD45.1 and CD45.2 antigens. Upon treatment, the BM cells were isolated and sorted for c-Kit and subsequently lysed for mRNA analyses.Mice: We used the PU.mulation . The hanmulation . As a homulation , with OCmulation , while tPU.1 gene. The data represent the % of the fully methylated DNA at the testis-specific H2B histone gene  relative to the input DNA. All data sets were compared using one-way ANOVA, Kruskal–Wallis two-tailed tests, a t-test , and Spearman’s correlation .Methyl DNA IP assay : The DNA was isolated using a DNeasy Blood and Tissue Kit . The amplicons evaluated via qPCR covered highly conserved and DNA-methylation-sensitive loci from distal enhancers (−16 kb upstream TSS) up to the +19.5 kb locus within the ® Lysis reagent  and evaluated on a Nano-Drop ND1000 Spectrophotometer . A High-Capacity cDNA Reverse Transcription Kit supplemented with miR-specific primers  produced a cDNA template for PCR on LightCycler Version 1.5.0.SP3 software with a 480 real-time PCR system  in 384-well plates. A run consisted of 40 cycles of 95 °C for 15 s and 60 °C for 1 min. For mRNA analyses, the TaqMan probe library  was used. For immunoblotting, BM cells were lysed in a RIPA buffer with protease and phosphatase inhibitors . Denatured cell lysates were run on gradient 8–16% Mini-PROTEAN TGX Stain-Free protein gels in a Mini-Protean Electrophoresis system and dry-blotted onto a PVDF membrane using a Trans-Blot Turbo transfer system . The PVDF membrane was blocked for 1 h in 5% non-fat milk in 1 × xTBS/0,1% Tween-20 (TBST) and incubated with primary antibodies: PU.1  and β-actin  O/N at 4 °C. Horseradish-peroxidase-conjugated secondary antibodies  were used to visualize bands using Westar Supernova ECL substrate .Expression: The RNA was isolated using the QIAzolure/ure). However, prolonged CEL treatment is unable to maintain this effect, and the compensatory upregulation of miR-155 results in accentuated myeloproliferation and splenomegaly. Thus, blocking miR-155 with AM155 represents a way to maintain the enhanced and pro-differentiation role of PU.1 in myeloid progenitors in the long term. Furthermore, we showed that AZA was also able to increase PU.1 levels at the same time that this treatment additionally prolonged the survival of mice with AML. Thus, we also indirectly confirmed that PU.1 is a very important obstacle in the pathogenesis of AML and that the hypermethylation of its promoter may play a role in the AML phenotype. By combining the aforementioned approaches, i.e., using AZA, CEL, and AM155, we were able to effectively inhibit AML growth in vitro and in vivo in CDX mice. Thus, we believe that the derepression of PU.1 protein levels and the inhibition of cell growth and survival may be modulated via the combined modification of the transcriptional level of the PU.1 gene and post-transcriptionally via inhibiting the miR-155 level.We have shown that the inhibition of the transcription factor MYB can increase PU.1 levels and induce its transcriptional program in a mouse model of AML (PU.1


Fluorescence lifetime imaging (FLIM) is a powerful tool that provides unique quantitative information for biomedical research. In this study, we propose a multi-layer-perceptron-based mixer (MLP-Mixer) deep learning (DL) algorithm named FLIM-MLP-Mixer for fast and robust FLIM analysis. The FLIM-MLP-Mixer has a simple network architecture yet a powerful learning ability from data. Compared with the traditional fitting and previously reported DL methods, the FLIM-MLP-Mixer shows superior performance in terms of accuracy and calculation speed, which has been validated using both synthetic and experimental data. All results indicate that our proposed method is well suited for accurately estimating lifetime parameters from measured fluorescence histograms, and it has great potential in various real-time FLIM applications. Curve-fitting methods have been commonly used for FLIM analysis, including the nonlinear least-square method (NLSM) [Fluorescence lifetime imaging (FLIM) is a powerful technique in biomedical research ,2, such 2+, O2) ,4,5,6,7.2+, O2) ,9. Fluor2+, O2) ,11. Amon2+, O2)  has been2+, O2) . TCSPC r2+, O2) :(1)yt=∑id (NLSM) , maximumd (NLSM) , Bayesiad (NLSM) , and Lagd (NLSM) . Howeverd (NLSM)  and the d (NLSM) . The phad (NLSM) ,22. CMM d (NLSM) ,23 offerd (NLSM) .Deep learning (DL) techniques have been proven promising for FLIM analysis ,25. It iWe proposed a new MLP-based DL algorithm to address the above challenges. Our algorithm adopts the MLP-Mixer  to proviThe idea behind the mixer is to separate the per-location (channel-mixing) operations and cross-location (token-mixing) operations. The skip connections can avoid gradient vanishing problems, leading to faster convergence and better performance. Our network has advantages with a light-weighted structure. The mixer relies on basic matrix multiplication routines, which require fewer hardware resources, including smaller memory size and less power consumption. It is much faster without sophisticated matrix operations. We can quickly implement hardware-friendly MLP neural networks in field-programmable gate array (FPGA) devices and integrate them into a wide-field SPAD FLIM system. In the training process, we use one block for the MLP-Mixer, in which the dimension of the token and channels are 16, and the patch size is 10.A is the amplitude of the underlying fluorescence decay, i-th fraction ratio, i-th lifetime component, and IRF(·) is the FLIM instrument response function, approximated by a Gaussian function with FWHM = 167 ps. The asterisk yt=IRIn most FLIM applications, signals’ underlying decay model is unknown; it is often valuable for determining average lifetimes rather than lifetime components, which are suspicious for biological interpretation. Amplitude-weighted average lifetimes can estimate FRET efficiency and access dynamic quenching behaviors, which are of great interest . TherefoX is the input and Y is the corresponding label of the signal. F is the mapping function with the network parameters Θ, and N is the batch size of the training dataset. The optimizer uses the Adam algorithm with a learning rate of 1 × 10−4 in the standard back-propagation [The proposed neural network was deployed in PyTorch 1.0 . The lospagation . The tra−4, indicating that the network is well trained as the estimated lifetimes are close to their ground truths. Because of the simple network structure, the number of hyperparameters is only 26,081. The training time is half an hour on a typical desktop using a central processing unit (CPU).The FWHM of IRFs has a Gaussian distribution with a mean value of 167 ps and a standard deviation of 60 ps. The training batch size is 200, and 20% of the total datasets are used for validation. An early stop callback with 20 patients is included to prevent overfitting. All the datasets are normalized to 1 before being fed into the neural network. n is the number of simulated decay curves. The MLP-Mixer was first evaluated on synthetic monoexponential signals with lifetimes of 1 and 4 ns. The photon counts range from 100 to 10,000, with 20 to 40 dB SNRs. We define the relative bias as Bias = |We also evaluate our method on histograms following a biexponential decay model. The short and long lifetime components, Dye solutions and plant cell samples were used for experimental validation. FLIM datasets were obtained with a commercial two-photon FLIM system.Rhodamine B and 6G saturated aqueous solutions were placed on a glass slide with coverslips. The FLIM system consists of a confocal microscope  and a TCSPC card . The femtosecond Ti: sapphire laser  has an excitation wavelength of 800 nm and a pulse width of less than 200 fs with an 80 MHz repetition rate. The bin width of TCSPC is 0.039 ns, and each measured histogram contains 256-time bins. The emission light passes through a 500–550 nm bandpass filter and then is collected by a 60× water-immersion objective lens (N.A = 1.0). We experimented on rhodamine 6G and rhodamine B at different SNRs to mimic low-light conditions. The results are shown in Convallaria majalis cell samples. The sample was measured with ‘high photon counts’ (HPC) at a 30 s acquisition time, ‘middle photon counts’ (MPC) at a 15 s acquisition time, and ‘low photon counts’ (LPC) at a 3 s acquisition time. We further evaluated the MLP-Mixer on Convallaria majalis cells. The results also suggest the MLP-Mixer’s excellent performance and robustness. Due to its simple architecture, it has the potential to be implemented in FPGA devices to accelerate the analysis for real-time FLIM applications.In summary, we present the design and training of the MLP-Mixer for FLIM analysis. The proposed algorithm has a simple architecture, which is easy to implement and hardware friendly. The simulated FLIM data analysis shows that the MLP-Mixer performs better, especially for small lifetime components. The trained model was then employed to analyze two-photon FLIM images of rhodamine 6G, rhodamine B, and


To calculate and evaluate postimplant dosimetry (PID) with CT-MR fusion technique after brain tumor brachytherapy and compare the result with CT-based PID. 125I) seed implantation entered this preliminary study for PID evaluation. Registration and fusion of CT and MR images of the same patients were performed one day after operation. Seeds identification and targets delineation were carried out on CT, MR, and CT-MR fusion images, each. The number and location of seeds on MR or CT- MR fusion images were compared with those of actually implanted seeds. Clinical target volume (CTV) and dosimetric parameters such as %D90, %V100 and external V100 were measured and calculated. In addition, the correlation of the fusion to CT CTV ratio and other factors were analyzed.  16 brain tumor patients received MR-guided intervention with Iodine-125 . The difference between reference seeds numbers and truly extracted MR seeds numbers was statistically significant . All dosimetric parameters showed significant differences between the two techniques (p <0.05). The mean CTV delineated on fusion images was 34.3 ± 33.6, smaller than that on CT images. The mean values of external V100, %V100 and %D90 on fusion images were larger than those on CT images. Correlation analysis showed that the fusion-CT V100 ratio was positively and significantly correlated with the fusion-CT volume ratio. 125I brain tumor brachytherapy dosimetry when compared to CT-based PID and merits further research to establish best-outcome protocols. This preliminary study indicated that CT-MR fusion-based PID exhibited good accuracy for  Therefohe brain . There hhe brain . Dosimethe brain , 9. Edemhe brain , while the brain . The majThe fusion of CT and MR images provides the potential for achieving anatomic definition by MR and seed detection by CT at the same time . This me125I brachytherapy for brain tumor from 2014 [125I seeds  by a specified doctor. Every case received a prescribed dose (PD) of 120Gy with mono-brachytherapy of 125I seeds. We summarized reasonable definition of PD from literature [This retrospective study received approval from our institutional review board. We have utilized MR in guidance of biopsy procedures and rom 2014 . Sixteenterature , 17. HerThe mainly interventional suite comprised of a 1.0-Tesla open MRI scanner . Real-time images were viewed by an on-site radiofrequency-shielded liquid crystal monitor in operation room. During operation, the number and position of seeds were confirmed and recorded as criterion to calculate accuracy of post-implanted seed extraction in CT or CT-MR fusion images. T1- or T2-weighted, turbo spin echo MR images with an axial slice thickness/gap of 3 mm/0 mm were acquired using a four-element head array coil  immediately after implantation achieved.CT scan for PID was completed one day after implantation on a DSCT unit . The images collecting parameters included 0.5-s tube rotation, 16-cm axial volumetric scanning range, 120-kVp tube voltage, 1-mm slice thickness and 0-mm interval. The images were reconstructed into 3-mm slice thickness and an interval of 0-mm, which is same as MR images. Both CT and MR scanning data were stored in the picture archiving and communication system (PACS).The ways of correlating CT and MR images are image registration and fusion. The purpose of image fusion is to infiltrate the seed positions highlighted on CT images into MR images. This process was performed using Syngo.via client 3.0 (Siemens) software. The requested data sets (including T1 or T2 MR image and reconstructed CT image data set) were retrieved from PACS via the network and were imported into Singo. These image data sets were input into a common coordinate system. The MR image data set was used as a reference, and CT image data set was reoriented and registered to the MR coordinate system. Some seed locations and anatomic structures which can be distinctly distinguished in both CT images and MR images acted as co-registration marks. By using manual rotation and movement in all the three spatial directions and corrections of a patient position, the same locations of co-registration marks in CT and MR image data sets completely coincided with each other. Once fusion is completed the fusion images were stored to PACS.The number and position of actually implanted seeds for every case were determined as reference seed coordinates by a series of procedures. Specifically, an operator informed the needle path and the number of seeds in each needle. Meanwhile, images along the needle direction were reconstructed with 1-mm slice thickness CT images . The invFor every case, 2 DICOM modalities (MR images and CT-MR fusion images) were downloaded from PACS and transferred to a treatment planning system  for PID verification, which include seed extraction, target contouring and dosimetry analysis, respectively. The interval between processing of the two modalities was more than 1 month to avoid any information of one image that may influence delineation and identification of the other. All delineation and identification were performed by a specific investigator (MZ). Clinical target volume (CTV) was determined by outward adding 5 mm on GTV. Here we defined primary dosimetric parameters such as %D90, %V100, and external V100 in the way of Kristina L. et al.  All dosit-test. Correlation analysis for the fusion to CT CTV ratio and other factors were calculated by SPSS 19 statistical software, and p <0.05 was statistically significant.Information about seeds, volume and dosimetry based on CT, MR, and CT-MR fusion images for every case was summed up and analyzed. Data about CTV, external V100, %D90 and %V100 are calculated and listed. Then volumetric or dosimetric ratios of CT-MR fusion images to CT images were estimated. The CT-MR fusion images were compared with CT images for each volume and dosimetric parameters by performing the two-sided paired The numbers of reference seeds ranged from 6 to 57, determined by their recorded number when they were implanted. Seed activities were same as 0.60 mCi at the time they were implanted. The numbers of fusion seeds were not significantly different compared with those of reference seeds . The difference between reference seeds numbers and true seeds numbers extracted on MR images was statistically significant . The differences of seeds numbers among the PID techniques are summarized in The main target dosimetric parameters, PTV, External V100, %D90 and %V100 were calculated and compared between CT- and fusion- based PID . All dosfusion/CT) was (0.85±0.10). Correlation analysis showed that the fusion-CT V100 ratio was positively and significantly correlated with the fusion-CT volume ratio , and fusion-CT D90 ratio was inversely and significantly correlated with the fusion-CT volume ratio . Total V100 and implanted seed number both showed low and non-significant correlation with the fusion-CT CTV ratio . As time passed, the volume of tumor becomes smaller and the seeds become more and more huddled together. Therefore isolating and identifying each seed becomes difficult. The other reason is related with seed coordinates relative to axial images. The standard axial tomography often does not coincide with the seed's long axis, which leads to an object possibly appearing on more than one slice and be regarded as two to three seeds. The images reconstructed along the needle path can display a complete seed on one slice .On MR images, if the signal intensity and size of the anatomical structure is the same as that of the seed, it is likely to be regarded as a “false seed”. If the seed position is close to the anatomical structure, which appears void signal, it is often thought a “missing seed” . In our The advantages of MR imaging include: (i) no ionizing radiation; (ii) excellent soft-tissue contrast . It is aMR imaging is good at anatomic discrimination but ineffective for seed confirmation. Complementarily, CT imaging can clearly show seeds, but it is not readily able to distinguish the boundary between normal brain tissues and tumors , 29. Thefusion/CT) and fusion/CT dosimetry ratios (V100fusion/CT and D90Fusion/CT). Therefore, we can infer fusion-based dosimetry to be superior to CT-based dosimetry. However, CT-MR fusion-based methods are costly and inconvenient because it requires both CT and MR scans. In addition, further research considering the estimation of accuracy of the therapeutic curve range is essential [CTVs produced by CT imaging were on average 15% larger than those produced by CT-MR fusion imaging. V100 also supported this fact. Accordingly, %V100 and %D90 on dose-volume histogram produced by CT imaging are different from those produced by CT-MR fusion imaging . Larger ssential  and is assential . Anotherssential , greaterssential . Here wessential , 37. Thessential .125I brain tumor brachytherapy than CT-based PID. Given the advantage of MR-based tissue definition and CT-based seed appearance, further study of CT-MR fusion-based dosimetry is warranted.CT-MR fusion-based PID exhibited greater accuracy in